Modulation of the transglycosylation activity of plant family GH18 chitinase by removing or introducing a tryptophan side chain

Abstract

Edited by Miguel De la RosaKeywords: Chitinase Transglycosylation Chitin oligosaccharide Tryptophan residue Crystal structure Cycas revoluta a b s t r a c t Transglycosylation (TG) activity of a family GH18 chitinase from the cycad, Cycas revoluta, (CrChiA) was modulated by removing or introducing a tryptophan side chain. The removal from subsite +3 through mutation of Trp168 to alanine suppressed TG activity, while introduction into subsite +1 through mutation of Gly77 to tryptophan (CrChiA-G77W) enhanced TG activity. The crystal structures of an inactive double mutant of CrChiA (CrChiA-G77W/E119Q) with one or two N-acetylglucosamine residues occupying subsites +1 or +1/+2, respectively, revealed that the Trp77 side chain was oriented toward +1 GlcNAc to be stacked with it face-to-face, but rotated away from subsite +1 in the absence of GlcNAc at the subsite. Aromatic residues in the aglycon-binding site are key determinants of TG activity of GH18 chitinases.

Chitinase: diversity, limitations, and trends in engineering for suitable applications

“…The former inactivated TG activity while the later enhanced TG activity. This study demonstrated the importance of the side chain of aromatic residues to TG activity [ 86 ]. TG was improved by altering the substrate-interaction sites involving residues in the catalytic center, catalytic cleft, and solvent exposed region of S. proteamaculans ChiD ( Sp ChiD).…”

“…TG takes place when retaining glycosidases trigger the transfer of a glycosidic residue (instead of a nearby nucleophilic water molecule in hydrolysis) from a donor to an acceptor yielding longer chain COS with high DP ( Figure 8 C) [ 24 ]. TG occurring during hydrolysis may be undesirable because it interrupts bond cleavage efficiency and causes misevaluation of hydrolytic activity because of the production of unexpected enzymatic products [ 86 ]. Serratia proteamaculans ChiD ( Sp ChiD) was found to exhibit ‘transglyco-hydrolytic’ activity with a hyper-TG activity on DP2 to DP6 substrates, thus generating long chain COS of DP up to 13 and a hydrolytic property that breaks down DP8 and DP10 TG products to DP2 and DP4 COS respectively [ 24 ].…”

“…However, their manner of chitin degradation and nature of COS produced in terms of degrees of polymerization, de-N-acetylation and acetylation, is highly varied [ 110 ]. For instance, certain chitinases interact with chitin in a hydrolytic manner to yield low molecular weight COS [ 34 , 114 ], while some utilize a transglycosylating mechanism for the addition of oligomeric units to yield longer chain oligomers [ 24 ] and others may have a combination of both mechanisms [ 86 ]. Therefore, the enzymatic degradation of crystalline chitin to give forth COS is not economically feasible because it might require an industrially complicated process involving the use of a combination of chitinases that are costly and not readily available [ 110 ].…”

Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida

“…is oriented in a way that a sugar ring can be stacked at subsite +3. Such tight substrate recognition at "plus" subsites is known to be a key determinant of transglycosylation activity in other glycoside hydrolases [50]. The tight substrate recognition also indicates that binding of M3 is stabilized at subsites +1, +2, and +3.…”

A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity

“…The reaction mechanism of GH 18 family enzymes is a retaining mechanism (catalysis leads to the retention of the anomeric configuration), whereas GH 19 enzymes use an inverting catalytic mechanism, resulting in anomeric inversion [8]. Plant chitinases are traditionally divided into different classes, and GH family 18 members are subdivided into the classes III and V. While there are numerous reports on class III chitinases [9], only three class V chitinases have been characterized, namely NtChiV from tobacco (Nicotiana tabacum) [10][11][12][13][14], AtChiC from Arabidopsis thaliana [15,16] and CrChiA from the gymnosperm Cycas revoluta [17][18][19][20]. Crystal structures for these three enzymes have been solved recently.…”